Multichannel Impedance Cytometry Downstream of Cell Separation by Deterministic Lateral Displacement to Quantify Macrophage Enrichment in Heterogeneous Samples

Abstract

cell types to exhibit subpopulations. [1] Heterogeneity arising from the phenotypic plasticity of immune, cancer and stem cells that serve multiple biological functions, [2] some with competing functional roles is important to quantify, since their balance regulates emergence of several diseases [3] and therapeutic outcomes. Hence, specific cellular subpopulations need to be enriched for quantifying their functional role and identifying disease markers. [4,5] While this is performed effectively by flow cytometry after fluorescent staining [6] or magnetic functionalization [7] of characteristic surface proteins, followed by fluorescent or magnetic activated cell sorting, the sample preparation is time consuming, requires costly chemicals, and introduces selection bias. Additionally, these operations are done off-chip, which causes sample loss, dilution and limits the enrichment level possible for fractional subpopulations. Furthermore, characteristic cell surface markers are often not available for biological functions, such as cancer metastasis, [8] stem cell differentiation lineage, [9] and immune cell activation. [10] Complementary approaches to identify cell phenotypes based on biophysical differences [11] in size, [12] shape, [13] deformability [14] and electrical properties [15] are emerging, but multiparametric approaches for high dimensional identification of The integration of on-chip biophysical cytometry downstream of microfluidic enrichment for inline monitoring of phenotypic and separation metrics at single-cell sensitivity can allow for active control of separation and its application to versatile sample sets. Integration of impedance cytometry downstream of cell separation by deterministic lateral displacement (DLD)for enrichment of activated macrophages from a heterogeneous sample is presented, without the problems of biased sample loss and sample dilution caused by off-chip analysis. This requires designs to match cell/particle flow rates from DLD separation into the confined single-cell impedance cytometry stage, the balancing of flow resistances across the separation array width to maintain unidirectionality, and the utilization of co-flowing beads as calibrated internal standards for inline assessment of DLD separation and for impedance data normalization. Using a heterogeneous sample with un-activated and activated macrophages, wherein macrophage polarization during activation causes cell size enlargement, on-chip impedance cytometry is used to validate DLD enrichment of the activated subpopulation at the displaced outlet, based on the multiparametric characteristics of cell size distribution and impedance phase metrics. This hybrid platform can monitor the separation of specific subpopulations from cellular samples with wide size distributions, for active operational control and enhanced sample versatility.

Dielectrophoretic enrichment of live chemo-resistant circulating-like pancreatic cancer cells from media of drug-treated adherent cultures of solid tumors

“…6A(i) ), which likely arises from necrotic cells released from the adherent culture under drug treatment. Using a DLD array designed for a cut-off size ( D C ) of 15 μm, 49 we would expect that most of the live cells would be collected in the displaced outlet and most of the dead cells would be collected in the zigzag outlet. While this is indeed the case based on Fig.…”

Rapid High-Throughput Isolation and purification of Chicken Myoblasts Based on Deterministic Lateral Displacement Microfluidic Chips

“…Deterministic lateral displacement (DLD) is a widely used technique in passive separation and has the advantages of simple structure, ease of processing, and high separation ability. It is extensively used for the isolation of a variety of cell types [14–17], especially human blood and tumor cells [18–20].…”